Figure 2: Adult CPCs Produce Functional Cardiomyocytes In Vitro and In Vivo

<b>(A)</b> Expression of calcium handling proteins (<i>CAV1.2</i>, <i>RYR2</i>, <i>SERCA2B</i>) in proliferating CPCs (Expansion; <b>black</b>) and in adult CPCs exposed to DLL1 <b>(gray)</b>, and differentiated in the absence or presence of DAPT. Data represent mean ± SEM; *p &lt; 0.05 as compared with proliferating CPCs; ‡p &lt; 0.05 as compared with differentiating unstimulated (None; <b>white</b>) CPCs in absence of DAPT (n = 4). <b>(B)</b> Cytosolic Ca²⁺ signals were recorded in adult CPCs in expansion <b>(upper panels)</b>, and in differentiating CPCs following exposure or not to DLL1 in the presence of DAPT. Cells were either evaluated under resting conditions or electrical stimulated (10 s at 0.5 Hz). Scale bar: 50 μm. Representative tracing of normalized Ca²⁺ changes obtained following induction of pacing <b>(arrow)</b>. <b>Colored lines</b> correspond to regions of recording as indicated in <b>B</b>. Images depicted in <b>B</b> were taken at the indicated time <b>(*)</b>. See Videos 1 and 2. <b>(C)</b> Quantification of calcium transient parameters. Data represent mean ± SEM; *p (white) CPCs in absence of DAPT (n = 55 cells per quantification). <b>(D)</b> Analysis of h-LAMIN <b>(red)</b> and α-ACTININ expression <b>(green)</b> in human CPC-derived cardiomyocytes in SCID mouse heart sections by immunostaining. DLL1-stimulated CPCs were injected in neonatal SCID mice. Mice were injected with DAPT or vehicle alone. Nuclei were stained with DAPI <b>(blue)</b>. Scale bars: 20 μm. <b>(E)</b> High magnification of human hLAMIN^POS α-ACTININ^POS cardiomyocytes in the mouse myocardium. Scale bars: 20 μm. <b>(F)</b> Quantification of human hLAMIN^POS cardiomyocyte patches in SCID mouse hearts. Data represent mean ± SEM; *p &lt; 0.05 as compared with control group (unstimulated CPCs [None]; No DAPT administration); ‡p &lt; 0.05 as compared with the group of animals injected with CPCs exposed to DLL1 <b>(gray)</b>, but not injected with DAPT (minimum 3,000 cells in 40 different fields at a magnification of 40× per quantification, 3 animals per condition). <b>(G)</b> Confocal analysis and tridimensional reconstruction of a representative patch of human cardiomyocytes in the mouse myocardium. <b>(a)</b> Confocal image of a representative patch. Scale bars: 20μm. <b>(b and c)</b> Tridimensional reconstruction of the representative human cardiomyocyte depicted in <b>a</b>. hLAMIN <b>(red)</b>, α-ACTININ expression <b>(green)</b>, DAPI <b>(blue)</b>. Scale bar: 5 μm. <b>(d and e)</b> High magnifications of areas indicated in <b>b and c</b>. Scale bar: 20 μm. Abbreviations as in Figure 1.